Special Methods

In a detailed study of a purified cellulase, additional details about the enzyme activity can contribute to the characterization and classification of the enzyme. Several techniques will be discussed that have been developed for determination of some important factors.

1. Inverting or Retaining—NMR Method (22, 23)

During the course of hydrolysis, the stereochemistry of the anomeric carbon formed is dependent on the structure and topology of the enzyme active site. Utilizing proton NMR, the hydrolysis of reduced cellodextrins (i.e., cellohexaitol) can be monitored for the formation of a- or ^-anomers. The enzymes can then be classified as inverting or retaining based on the product stereochemistry. Dividing cellulases based on the primary sequences into families shows that there is very strong relationship between the family and the stereochemical course of hydrolysis.

2. Cellulose Chain End Determination Method (24)

Until recently it was believed that the mechanism of hydrolysis for exocellulases (cellobiohydrolases) was solely from the nonreducing end of the cellulose molecule. In 1996, Barr et al. (24) used 18O and 14C labeling of cellooligosaccharides in conjunction with ion-spray mass spectrometry to determine the precise position and direction of hydrolysis of five different exocellulases from Thermonospora fusca and Trichoderma reesei. Their results support the conclusion that there are two different classes of exocellulases each working from different ends of the cellulose molecules.

3. Dialysis Reactor Cell Method (25)

Synergism is an interesting observation shown with cellulases and with other enzymes, such as amylase, that have insoluble substrates and multiple types of enzymes working on the same substrate. Determination of synergism is difficult, especially using a native substrate. Baker et al. (25) developed a microdialysis device for studying synergism using native or pretreated substrates, under conditions in which product inhibition is minimized. The reactor cell is loaded with substrate and enzymes and held under constant temperature. The cell is continually flushed with fresh buffer, and the eluent is collected and assayed using HPLC sugar analysis.